The research plan involves studying three distinct mutations in the fusion gene P210 BCR/ABL, the molecular hallmark of the Philadelphia Chromosome (Ph') of Chronic Myelogenous Leukemia (CML). These mutations involve areas of BCR/ABL recently shown to have potentially important roles in how it causes transformation in several in vitro systems. The three mutations include: (1) deletion of the SH2 domain, a conserved regulatory region in c-Abl and many other proteins, important in signal transduction between proteins phosphorylated on tyrosine and mediators of proliferation and differentiation; (2) a point mutant in a region of BCR (Y177F) shown to be an important link between Ras and BCR/ABL in cellular transformation; (3) deletion of a region in the carboxy-terminus of c-Abl that has recently been shown to mediate the interaction of Abl with the F-actin cytoskeleton. Much remains to be learned about whether these regions are important in transformation of hematopoietic cells in tissue culture and in vivo. The basic res arch design to accomplish these goals consists of two phases, the first involves studying these mutations in tissue culture, and the second will be to use the insight gained in vitro to establish in vivo relevance by studying their effects in murine bone marrow transplant experiments. In the first part, these mutant constructs will be introduced into the murine hematopoietic factor-dependent cell liens Ba/F3 (lymphoid), and FDCP-1 (myeloid) by retroviral gene transfer and electroporation, respectively. Outcome will be assessed by how these P210BCR/ABL mutants compare in their ability to transform these cells to growth factor independence compared to wild type P210. In the second phase these mutants will be introduced into murine bone marrow cells by a Rat-1 fibroblast retroviral packaging cell lin. After infection, the marrow cells will be used to reconstitute syngeneic lethally irradiated recipients, and the incidence and leukemic phenotype which results will be compared to parallel P210 controls. These studies will provide important insight into the pathogenesis of BCR/ABL- related malignancies, and perhaps someday benefit patients suffering from these diseases.
